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Proteomics analysis of mass-limited samples has become increasingly important for understanding biological systems in physiologically relevant contexts such as patient samples, multicellular organoids, spheroids, and single cells. However, relatively low sensitivity in top-down proteomics methods makes their application to mass-limited samples challenging. Capillary electrophoresis (CE) has emerged as an ideal separation method for mass-limited samples due to its high separation resolution, ultralow detection limit, and minimal sample volume requirements. Recently, we developed "spray-capillary", an electrospray ionization (ESI)-assisted device, that is capable of quantitative ultralow-volume sampling (e.g., pL-nL level). Here, we developed a spray-capillary-CE-MS platform for ultrasensitive top-down proteomics analysis of intact proteins in mass-limited complex biological samples. Specifically, to improve the sensitivity of the spray-capillary platform, we incorporated a polyethylenimine (PEI)-coated capillary and optimized the spray-capillary inner diameter. Under optimized conditions, we successfully detected over 200 proteoforms from 50 pg of E. coli lysate. To our knowledge, the spray-capillary CE-MS platform developed here represents one of the most sensitive detection methods for top-down proteomics. Furthermore, in a proof-of-principle experiment, we detected 261 ± 65 and 174 ± 45 intact proteoforms from fewer than 50 HeLa and OVCAR-8 cells, respectively, by coupling nanodroplet-based sample preparation with our optimized CE-MS platform. Overall, our results demonstrate the capability of the modified spray-capillary CE-MS platform to perform top-down proteomics analysis on picogram amounts of samples. This advancement presents the possibility of meaningful top-down proteomics analysis of mass-limited samples down to the level of single mammalian cells.
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Eletroforese Capilar , Proteômica , Eletroforese Capilar/métodos , Proteômica/métodos , Humanos , Escherichia coli/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas/métodosRESUMO
Sevoflurane has been reported to have anti-tumorigenic effects in glioma. Circ_0000215 was found to play vital functions in the pathological progressions of glioma. However, whether circ_0000215 mediates the inhibitory effects of sevoflurane on glioma cells remains unclear. In vitro assays were performed using cell counting kit-8, flow cytometry, transwell and Western blot assays. The expression levels of circ_0000215, microRNA (miR)-1200 and NCR3LG1 (Natural Killer Cell Cytotoxicity Receptor 3 Ligand 1) were detected using quantitative real-time polymerase chain reaction (qRT-PCR) and/or Western blot. The dual-luciferase reporter assay and pull-down assay were used to investigate the relationship between miR-1200 and circ_0000215 or NCR3LG1. In vivo assay was conducted using xenograft nude mice model. In vitro assays suggested that sevoflurane repressed glioma cell proliferation, metastasis and induced apoptosis as well as hindered tumor growth in vivo, which were reversed by circ_0000215 overexpression. Mechanically, circ_0000215 was confirmed to directly target miR-1200, and NCR3LG1 was a target of miR-1200 in glioma cells. Importantly, circ_0000215 could regulate NCR3LG1 expression via miR-1200. Besides that, rescue assay suggested that circ_0000215 attenuated the inhibitory effects of sevoflurane on glioma cell growth and metastasis, which were reversed by miR-1200 overexpression or NCR3LG1 knockdown. Our study revealed that sevoflurane could suppress glioma tumorigenesis by regulating circ_0000215/miR-1200/NCR3LG1 axis, suggesting a new insight into the therapeutic potential of sevoflurane in glioma treatment.
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Neoplasias Encefálicas , Glioma , MicroRNAs , Animais , Antígenos B7/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Movimento Celular , Glioma/tratamento farmacológico , Glioma/metabolismo , Humanos , Camundongos , Camundongos Nus , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Circular/genética , Sevoflurano/farmacologia , Sevoflurano/uso terapêuticoRESUMO
In many areas of application, key objectives of chemical separation and analysis are to minimize the sample quantity while maximizing the chemical information obtained. Increasing measurement sensitivity is especially critical for proteomics research, especially when processing trace samples and where multiple measurements are desired. A rich collection of technologies has been developed, but the resulting sensitivity remains insufficient for achieving in-depth coverage of proteomic samples as small as single cells. Here, we combine picoliter-scale liquid chromatography (picoLC) with mass spectrometry (MS) to address this issue. The picoLC employs a 2-µm-i.d. open tubular column to reduce the sample input needed to greatly increase the sensitivity achieved using electrospray ionization (ESI) with MS. With this picoLC-MS system, we show that we can identify â¼1000 proteins reliably using only 75 pg of tryptic peptides, representing a 10-100-fold sensitivity improvement compared with the state-of-the-art liquid chromatography (LC) or capillary electrophoresis (CE)-MS methods. PicoLC-MS extends the limit of separation science and is expected to be a powerful tool for single cell proteomics.
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Peptídeos/análise , Proteômica , Cromatografia Líquida , Eletroforese Capilar , Células HeLa , Humanos , Espectrometria de Massas , Tamanho da Partícula , Análise de Célula Única , Propriedades de SuperfícieRESUMO
Separation speed and resolution are two important figures of merit in chromatography. Often, one gains the speed at the cost of the resolution, and vice versa. Scientists have employed short-packed columns for ultrafast separations but encountered challenges such as limited mobile phase velocity, extra-column effect caused band broadening, and column packing difficulty. We have recently demonstrated ultrahigh resolutions of narrow open tubular liquid chromatography (NOTLC); this allows us to trade some of the resolution for speed. In this work, we explored NOTLC for ultrafast LC separations. We used a 2.7 cm (effective length) narrow open tubular (NOT) column and showed a baseline separation of 6 amino acids in less than 700 ms. Ways to further increase the speed were discussed. Using short narrow open tubular (NOT) columns to perform ultrafast separation we overcame the challenges from using short packed columns. To demonstrate the feasibility of using this ultrafast separation technique for practical applications, we separated complex protein digests; peptides were nicely resolved in â¼1 min.
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The advancements in life science research mandate effective tools capable of analyzing large numbers of samples with low quantities and high complexities. As an essential analytical tool for this research, liquid chromatography (LC) encounters an ever-increasing demand for enhanced resolving power, accelerated analysis speed, and reduced limit of detection. Although theoretical studies have indicated that open tubular (OT) columns can produce superior resolving power under comparable elution pressures and analysis times, ultrahigh-resolution and ultrahigh-speed open tubular liquid chromatography (OTLC) separations have never been reported. Here we present experimental results to demonstrate the predicted potential of this technique. We use a 2 µm i.d. × 75 cm long OT column coated with trimethoxy(octadecyl)silane for separating pepsin/trypsin digested E. coli lysates and routinely produce exceptionally high peak capacities (e.g., 1900-2000 in 3-5 h). We reduce the column length to 2.7 cm and exhibit the capability of OTLC for ultrafast separations. Under an elution pressure of 227.5 bar, we complete the separation of six amino acids in â¼800 ms and resolve these compounds within â¼400 ms. In addition, we show that OTLC has low attomole limits of detection (LOD) and each separation requires samples of only a few picoliters. Importantly, no ultrahigh elution pressures are required. With the ultrahigh resolution, ultrahigh speed, low LOD, and low sample volume requirement, OTLC can potentially be a powerful tool for biotech research, especially single cell analysis.
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Aminoácidos/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Escherichia coli/química , Peptídeos/isolamento & purificação , Cromatografia Líquida de Alta Pressão/instrumentação , Misturas Complexas/química , Limite de Detecção , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos , Fatores de TempoRESUMO
In this work, we outline a signal amplification strategy using the coordination chemistry between Fe3+ and poly(glutamic acid) (PGA) for biosensing applications. The theoretical calculation based on density functional theory shows that PGA has a much higher binding affinity with Fe3+ than the other metal ions. Guided by this rationale, we prepare a PGA-mediated signal probe through conjugating PGA onto polystyrene (PS) nanoparticles to form a brushlike nanostructure for Fe3+ coordination. This PGA-PS brush (PPB) has a large loading capacity of Fe3+ with a number of 1.92 × 108 Fe atoms per nanoparticle that greatly amplifies the signals for assays in an enzyme-free way. Combined with ferrozine coloration-based readout, this PPB-mediated amplification is further applied for the enzyme-free immunoassay that shows an ultrahigh sensitivity for detection of microcystins-LR (12 pg/mL), a 5-fold enhancement compared with that of traditional enzyme-linked immunosorbent assay (ELISA) (60 pg/mL). In addition, the good stability, rapid response, and long shelf life make this enzyme-free amplification strategy a promising platform for point-of-care biosensing applications.
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Técnicas Biossensoriais , Compostos Férricos/química , Imunoensaio , Microcistinas/análise , Ácido Poliglutâmico/química , Água Potável/química , Humanos , Toxinas Marinhas , Nanopartículas/química , Sistemas Automatizados de Assistência Junto ao Leito , Poliestirenos/químicaRESUMO
Students' first-year academic success plays a critical role on their overall development in college, which implies the need to concentrate on identifying ways to improve students' first-year academic success. Different from most research on the subject, this study attempted to combine the sociological perspective of college impact with a psychological perspective to synthetically explore the causal relationship of specific types of self-concept and college involvement with academic success of medical students. A longitudinal study was conducted using 519 matriculates at a medical university in mainland China. We conducted the Cooperative Institutional Research Program freshmen survey and the Your First College Year survey to collect data of the pre-college and college academic and social self-concept, college involvement components, and some input characteristics. The academic success was measured by the first-year grade point average. A pathway analysis was conducted and showed the following results. Having high academic self-concept, being engaged in class and putting effort in homework or study directly contributes to increasing college achievement. Students' pre-college achievement and self-concept, faculty interaction, and homework involvement positively affected students' college academic self-concept development, which indirectly improved average grade point. These findings contribute to our understanding of a student's ability to interact with his or her collegiate environment and to experience academic success.
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Logro , Educação de Graduação em Medicina , Autoimagem , Estudantes de Medicina/psicologia , China , Avaliação Educacional , Feminino , Humanos , Estudos Longitudinais , Masculino , Inquéritos e Questionários , Adulto JovemRESUMO
BACKGROUND: Although medical education has developed rapidly in the last decade, and the National College Entrance Examination (NCEE) is used as the "gold standard" for admission to medical college in mainland China, there is a lack of literature regarding the influence of NCEE score and other factors on the academic performance of medical students. This study aimed to examine potential predictors of first-year grade point average (GPA) for medical students. METHODS: This study included 1,285 students who matriculated at a first-tier medical university in mainland China in 2011. The precollege motivational attitudes for each matriculate were investigated via questionnaire. A hierarchical linear model was fitted to regress first-year GPA on a 100-point scale on NCEE score and other student-level and major-level characteristics. RESULTS: NCEE score was a significant predictor of both within-major and between-major variation of first-year GPA for medical students. Majors with higher mean NCEE scores had higher mean GPAs, and higher GPAs were observed among those individuals with higher NCEE scores after controlling for major-level characteristics. First-year GPA differed by certain individual socio-demographic variables. Female students had a 2.44-higher GPA on average than did male students. NCEE repeaters had a 1.55-lower GPA than non-repeaters. First-year GPA was associated negatively with parental income but positively with academic self-concept. CONCLUSIONS: NCEE score is an important predictor of the first-year GPA of medical students, but it is not the sole determinant. Individual socio-demographic characteristics and major-level characteristics should be taken into account to understand better and improve the first-year GPA of medical students.
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Escolaridade , Estudantes de Medicina/estatística & dados numéricos , China/epidemiologia , Teste de Admissão Acadêmica , Avaliação Educacional , Feminino , Humanos , Estudos Longitudinais , Masculino , Faculdades de Medicina/normas , Faculdades de Medicina/estatística & dados numéricos , Fatores Sexuais , Fatores SocioeconômicosRESUMO
Interface strain significantly affects the band structure and electronic states of metal-nanocrystal-2D-semiconductor heterostructures, impacting system performance. While transmission electron microscopy (TEM) is a powerful tool for studying interface strain, its accuracy may be compromised by sample overlap in high-resolution images due to the unique nature of the metal-nanocrystals-2D-semiconductors heterostructure. Utilizing digital dark-field technology, the substrate influence on metal atomic column contrasts is eliminated, improving the accuracy of quantitative analysis in high-resolution TEM images. Applying this method to investigate Pt on MoS2 surfaces reveals that the heterostructure introduces a tensile strain of ≈3% in Pt nanocrystal. The x-directional linear strain in Pt nanocrystals has a periodic distribution that matches the semi-coherent interface between Pt nanocrystals and MoS2, while the remaining strain components localize mainly on edge atomic steps. These results demonstrate an accurate and efficient method for studying interface strain and provide a theoretical foundation for precise heterostructure fabrication.
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Cardiovascular diseases (CVDs) are the leading cause of death worldwide, and the prevalence of CVDs increases markedly with age. Due to the high energetic demand, the heart is highly sensitive to mitochondrial dysfunction. The complexity of the cardiac mitochondrial proteome hinders the development of effective strategies that target mitochondrial dysfunction in CVDs. Mammalian mitochondria are composed of over 1000 proteins, most of which can undergo post-translational protein modifications (PTMs). Top-down proteomics is a powerful technique for characterizing and quantifying all protein sequence variations and PTMs. However, there are still knowledge gaps in the study of age-related mitochondrial proteoform changes using this technique. In this study, we used top-down proteomics to identify intact mitochondrial proteoforms in young and old hearts and determined changes in protein abundance and PTMs in cardiac aging. METHODS: Intact mitochondria were isolated from the hearts of young (4-month-old) and old (24-25-month-old) mice. The mitochondria were lysed, and mitochondrial lysates were subjected to denaturation, reduction, and alkylation. For quantitative top-down analysis, there were 12 runs in total arising from 3 biological replicates in two conditions, with technical duplicates for each sample. The collected top-down datasets were deconvoluted and quantified, and then the proteoforms were identified. RESULTS: From a total of 12 LC-MS/MS runs, we identified 134 unique mitochondrial proteins in the different sub-mitochondrial compartments (OMM, IMS, IMM, matrix). 823 unique proteoforms in different mass ranges were identified. Compared to cardiac mitochondria of young mice, 7 proteoforms exhibited increased abundance and 13 proteoforms exhibited decreased abundance in cardiac mitochondria of old mice. Our analysis also detected PTMs of mitochondrial proteoforms, including N-terminal acetylation, lysine succinylation, lysine acetylation, oxidation, and phosphorylation. CONCLUSION: By combining mitochondrial protein enrichment using mitochondrial fractionation with quantitative top-down analysis using ultrahigh-pressure liquid chromatography (UPLC)-MS and label-free quantitation, we successfully identified and quantified intact proteoforms in the complex mitochondrial proteome. Using this approach, we detected age-related changes in abundance and PTMs of mitochondrial proteoforms in the heart.
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Top-down proteomics (TDP) identifies, quantifies, and characterizes proteins at the intact proteoform level in complex biological samples to understand proteoform function and cellular mechanisms. However, analyzing complex biological samples using TDP is still challenging due to high sample complexity and wide dynamic range. High-resolution separation methods are often applied prior to mass spectrometry (MS) analysis to decrease sample complexity and increase proteomics throughput. These separation methods, however, may not be efficient enough to characterize low abundance intact proteins in complex samples. As such, multidimensional separation techniques (combination of two or more separation methods with high orthogonality) have been developed and applied that demonstrate improved separation resolution and more comprehensive identification in TDP. A suite of multidimensional separation methods that couple various types of liquid chromatography (LC), capillary electrophoresis (CE), and/or gel electrophoresis-based separation approaches have been developed and applied in TDP to analyze complex biological samples. Here, we reviewed multidimensional separation strategies employed for TDP, summarized current applications, and discussed the gaps that may be addressed in the future.
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Flow injection chromatography (FIC) or sequential injection chromatography (SIC) is a low-pressure liquid chromatography technique that uses flow injection or sequential injection hardware. Due to the constraints of this hardware, the separation resolution is low; often no more than 3-5 components are resolved. We have recently demonstrated the excellent resolving power of narrow open tubular (OT) columns for various biomolecules, and only moderate elution pressures are needed to carry out these separations. In this paper, we incorporate a narrow OT column with FIC and construct an FIC system using a pressure chamber and two injection valves to implement gradient elution. The resultant system not only improves the resolution but also reduces the system cost. When we use the system to separate peptides from trypsin-digested cytochrome C, we can resolve dozens of peptides (with resolutions of 0.5 or greater) at a speed of 12 samples per hour. When we use this system to separate a mixture containing 3 amino acids, we can base-line resolve these compounds at a speed of 1800 sample per hour.
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Aminoácidos/isolamento & purificação , Análise de Injeção de Fluxo/instrumentação , Aminoácidos/química , Cromatografia Líquida/instrumentaçãoRESUMO
It is of great importance to propose effective methods to quantify interfacial interaction since it directly determines foulant adhesion and membrane fouling process in membrane bioreactors (MBRs). This study developed a radial basis function (RBF) artificial neural network (ANN) to predict the interfacial interactions with randomly rough membrane surface. The interaction data quantified by the advanced extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) approach were used as the training samples for the RBF networks. It was found that, the computing time consumption for the RBF network prediction was only about 1/50 of that for the advanced XDLVO approach under same conditions, indicating the high efficiency of the RBF ANN method. Meanwhile, the calculation accuracy of the method was acceptable to get reliable results. This study demonstrated the breakthrough of the fundamental methodology related with membrane fouling. The proposed RBF ANN method has broad application prospects in membrane fouling and interface behavior research.
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Incrustação Biológica , Reatores Biológicos , Redes Neurais de ComputaçãoRESUMO
Research focused on transforming growth factor ß (TGFß) signaling in osteoblast is gradually increasing, whereas literature is rare in terms of fluorosis. This work aimed to investigate how TGFß signaling participated in regulation of the osteoblast by different doses of fluoride treatment. Bone marrow stem cells (BMSCs) were developed into osteoblastic cells and exposed to 1, 4, and 16 mg/L F- with and without 10 ng/mL of TGFß. Cell viability and differentiation state of osteoblast under different settings were measured by means of cell counting kit and analysis of alkaline phosphatase (ALP) activity as well as formation of mineral nodules. Real-time PCR was utilized to test expression of ALP and Runt-related transcription factor 2 (Runx2) at gene level. The gene expression of TGFß signaling effectors was also investigated, such as TGFß receptors (TßRs), smad3, and mitogen-activated protein kinases (MAPK). Results demonstrated that fluoride treatment exhibited action on osteoblast viability and osteogenic differentiation and upregulated expression of TßR2, smad3, and MAPK in this process. Administration of TGFß strengthened ALP activity but attenuated formation of mineral nodules. Co-treatment of TGFß and low-dose fluoride increased ALP activity compared to same dose of single fluoride treatment, whereas it inhibited mineral nodule formation. Administration of TGFß reversed the suppression of high-dose fluoride on osteogenic differentiation of BMSCs. Taken together, studies revealed that TßR2 acted as a target for fluoride and TGFß treatment on BMSCs, and smad3 and MAPK were involved in the mechanism of fluoride regulating osteogenic differentiation. Together, our data indicated that TGFß receptor-mediated signaling through smad3 and MAPK was required for modulation of fluoride on osteoblast viability and differentiation, and activating TßR2-smad3 signaling pathway reversed suppression of osteoblasts differentiation by high dose of fluoride treatment.
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Diferenciação Celular/efeitos dos fármacos , Fluoretos/farmacologia , Osteoblastos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Masculino , Camundongos , Camundongos Endogâmicos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Fator de Crescimento Transformador beta/administração & dosagem , Fator de Crescimento Transformador beta/genéticaRESUMO
OBJECTIVE: The aim of this study was to explore the effect of miR-146b expression and variants on endometriosis and its associated pain symptom. MATERIALS AND METHODS: Genotyping and expression of miR-146b was performed on 74 endometriosis patients and 23 healthy controls. ESCs were subsequently co-cultured with peripheral blood (PB)-derived monocytes (PBMC)-driven macrophages. After overexpression and inhibition of miR-146b, cytokine production from the macrophages were determined by enzyme-linked immunosorbent assay (ELISA). Western blot were done to measure the regulation of IRF5 by miR-146b. RESULTS: We found that miR-146b expression was increased in PF supernatant and PF CD14 + monocytes/Macrophages of endometriosis patients, with endometriosis patients with pain (EPWP) showing higher miR-146b expression compared with the endometriosis patients without pain (EPNP). CT/CC genotype of miR-146b rs1536309 was associated with the risk of pain symptom of endometriosis. For the function studies, we found that miR-146b was involved in the negative regulation of inflammation through attenuating IRF5 expression. Macrophages from patients who carries CT/CC genotype of miR-146b rs1536309 showed decreasing miR-146b expression and enhancement of the ability of pro-inflammation. CONCLUSIONS: Our findings suggest an important role of miR-146b level and variants in endometriosis that helps to regulate the process of endometriosis and its associated pain.
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Endometriose/genética , MicroRNAs , Dor/genética , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Fatores Reguladores de Interferon , Macrófagos/metabolismo , Células Estromais/metabolismo , Inquéritos e QuestionáriosRESUMO
We have recently obtained extraordinarily high efficiencies and sharp peaks using narrow open tubular (OT) columns for liquid chromatographic separations. On-column focusing is commonly observed in liquid chromatography, but this effect alone could not satisfactorily explain the sharpness of these peaks. In this work we investigated the reasons that could have led to the peak sharpness. We hypothesized initially that analytes confined in a narrow OT column might have significantly reduced analyte diffusivities and the reduced diffusivities consequently resulted in the peak sharpness. This hypothesis was invalidated immediately after we measured the diffusion coefficients and did not notice any noticeable diffusivity increases of the analytes inside such columns. We then designed an experiment and revealed a "re-focusing effect". Investigation of this re-focusing effect eventually led us to the observation of a gradient focusing caused by the composition difference between the eluent and the sample matrix. It was this gradient focusing that had contributed primarily to the peak sharpness. On the basis of this insightful understanding, we further developed a simple and economic approach to perform pico-gradient narrow open tubular liquid chromatographic separations.
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We investigated the relationship between oxidative stress and osteoblasts viability in osteoblasts exposed to various concentrations of fluoride in this study. Primary calvarial osteoblasts from neonatal Kunming mice were cultured and subcultured to the third generation. Osteoblasts were incubated with sodium fluoride (0, 0.5, 1, 2, 4, 8, 12, and 20 mgF(-)/L) for 24, 48, and 72 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis showed cell viability significantly increased after osteoblasts exposed to low concentrations of fluoride (0.5 to approximately 2 mgF(-)/L) for 24 to approximately 72 h. Oxidative stress analysis showed that low concentration of fluoride excited lipid peroxidation in osteoblasts and increased activity of antioxidant enzymes in varying degrees. We demonstrated that changes of osteoblasts viability of the low-dose fluoride groups are different from those of high-dose fluoride groups; however, both low and high doses of fluoride caused active state of oxidative stress in osteoblasts, which suggesting that oxidative stress may be excited by the active osteoblasts viability induced by a low dose of fluoride.
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Osteoblastos/efeitos dos fármacos , Estresse Oxidativo , Fluoreto de Sódio/farmacologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Camundongos , Osteoblastos/metabolismoRESUMO
Studies that have focused on the role TGFß signaling plays in osteoclast activity are gradually increasing; however, literature is rare in terms of fluorosis. The aim of this study is to observe the role the TßR1/Smad3 pathway plays in fluoride regulating cellsosteoclast-like cells that are under the treatment of TGFß receptor 1 kinase. The RANKL-mediated osteoclast-like cells from RAW264.7 cells were used as osteoclast precursor model. The profile of miRNA expression in fluoride-treated osteoclast-like cells exhibited 303 upregulated miRNAs, 61 downregulated miRNAs, and further drew 37 signaling pathway maps by KEGG and Biocarta pathway enrichment analysis. TGFß and its downstream effectors were included among them. Osteoclast viability, formation and function were detected via MTT method, bone resorption pit and tartrate-resistant acid phosphatase (TRACP) staining, respectively. Results demonstrated that different doses of fluoride exhibited a biphasic effect on osteoclast cell viability, differentiation, formation and function. It indicated that a low dose of fluoride treatment stimulated them, but high dose inhibited them. SB431542 acted as TßR1 kinase inhibitor and blocked viability, formation and function of osteoclast-like cells regulated by fluoride. The expression of the osteoclast marker, RANK, and TßR1/Smad3 at gene and protein level was analyzed under fluoride with and without SB431542 treatment. Fluoride treatment indicated little effect on the RANK protein expression; however it significantly influenced TRACP expression in osteoclast-like cells. The stimulation of fluoride on the expression of Smad3 gene and phosphorylated Smad3 protein exhibited dose-dependent manner. SB431542 significantly impeded phosphorylation of Smad3 protein and TRACP expression in osteoclast-like cells that were exposed to fluoride. Our work demonstrated that TGFß signaling played a key role in fluoride regulating osteoclast differentiation, formation and function. It elucidated that TßR1/Smad3 pathway participated in the mechanism of biphasic modulation of osteoclast mode regulated by fluoride.
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Proteínas da Matriz Extracelular , Fluoretos/toxicidade , Osteoclastos/efeitos dos fármacos , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta , Animais , Benzamidas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dioxóis/farmacologia , Proteínas da Matriz Extracelular/antagonistas & inibidores , Proteínas da Matriz Extracelular/genética , Expressão Gênica/efeitos dos fármacos , Camundongos , MicroRNAs/genética , Osteoclastos/citologia , Osteoclastos/metabolismo , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Proteína Smad3/genética , Células-Tronco , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/genéticaRESUMO
Overexposure to fluoride from environmental sources can cause serious public health problems. Disrupted osteoblast function and impaired bone formation were found to be associated with excessive fluoride exposure. A massive analysis of microRNAs (miRNAs) was used to figure out the possible pathways in which fluoride affects osteoblast function. MC3T3-E1 cells were treated with 8 mg/L of fluorine for 7 days. Total RNA of cells was extracted, and their integrity and purity were tested. RNA samples were analyzed by using miRNA array, including miRNA labeling, hybridization, scanning, and expression data analysis to compare the profiling of miRNA expression between control and fluoride-treated group. Transcriptome analysis console and enrichment analysis calculated by miRSystem were used to predict target genes and collect miRNAs pathway maps. Forty-five upregulated and 31 downregulated miRNAs expression were found in the fluoride-treated group, and most of the verified miRNAs were mature. The KEGG pathway enrichment analysis searched out 36 pathways that scored more than 0.1. These pathways mainly included intracellular signaling, cytokines, metabolism, and cytoskeleton-related pathways. Among them, the Wnt, insulin, TGF-beta, hedgehog, VEGF, and notch pathways in osteoblasts were those mainly affected by fluoride treatment. These results have shown a number of higher level systemic pathways activated by overexposure of fluoride in osteoblastic cells and verified that fluoride affected the molecular crosstalk in the osteoblasts.
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Fluoretos/farmacologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , MicroRNAs/análise , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Animais , Linhagem Celular , MicroRNAs/biossíntese , RNA Mensageiro/análise , RNA Mensageiro/biossínteseRESUMO
We have developed a magnetic enzyme-linked immunosorbent assay (MELISA) based poly-L-lysine (PLL) mediated brushes on magnetic nanoparticles (MNPs) for detection of Escherichia coli O157:H7 in river water samples. In the MELISA, we couple PLL on the surface of magnetic nanoparticles to prepare the PLL brushes (MPLLBs). PLL with plentiful amine groups provides multi-binding sites to allow the binding of both horse radish peroxidase (HRP) and antibody (Ab) on the surface of the MPLLBs with high capacity. Compared with the conventional particles (the binding capacity of MNPs for HRP and Ab are 19µg/mg and 16µg/mg, respectively), MPLLBs have achieved an improvement of 43-fold and 24-fold in binding capacity for HRP (816µg/mg, protein µg per mg of MPLLBs) and Ab (387µg/mg), respectively. This multifunctional Ab-MPLLBs-HRP conjugate serves as not only an immune-carrier for magnetic enrichment but also an enzyme assembly for signal amplification system. Compared with the conventional ELISA (the detection of limit is 400 cfu/mL), MELISA shows enhanced sensitivity (8cfu/mL) and shortened the analysis time (within 2h) for the detection of Escherichia coli O157:H7 in river water sample, which provides an attractive candidate platform for the rapid and sensitive detection of pathogen in complex samples.